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212
Reports
combination of unilateral SO palsy and heterotopic pulleys can
produce a clinical pattern that resembles bilateral SO palsy,
with excessive excyclotorsion compared with a unilateral SO
palsy in the setting of normal EOM pulley position.
References
1. Demer JL, Miller JM, Poukens V, Vinters HV, Glasgow BJ. Evidence
for fibromuscular pulleys of the recti extraocular muscles. Invest
Opbthalmol Vis Sd. 1995;36:1125-1136.
2. Miller JM. Functional anatomy of normal human rectus muscles.
Vision Res. 1989;2S>:223-240.
3. Clark RA, Miller JM, Demer JD. Location and stability of rectus
muscle pulleys: muscle paths as a function of gaze. Invest Ophthalmol Vis Sci. 1997;38:227-240.
4. Demer JL, Miller JM, Rosenbaum AL. Effect of transposition surgery
on rectus muscle paths by magnetic resonance imaging. Ophthalmology. 1993;100:475-487.
Establishment and
Characterization of a Retinal
Muller Cell Line
Vijay P. Sarthy,1 Sevan J. Brodjian,1
Kamla Dutt,2 Breandan N. Kennedy?
Randall P. French,1 and John W. Crabb5
Primary cultures of Miiller cells have proven
useful in cell biologic, developmental, and electrophysiological studies of Miiller cells. However, the limited
lifetime of the primary cultures and contamination from
non-neural cells have restricted the utility of these cultures. The aim of this study was to obtain an immortalized
cell line that exhibits characteristics of Muller cells.
PURPOSE.
METHODS. Primary Muller cell cultures were
prepared from
retinas of rats exposed to 2 weeks of constant light. Cells
were immortalized by transfection with simian virus 40.
Single clones were obtained by repeatedly passaging cells
using cloning wells. Immunocytochemical and immunoblotting studies were carried out with glial nbrillary acidic
protein (GFAP)-specific and cellular retinaldehyde-binding
protein (CRALBP)-specinc antibodies. Transient transfections with CRALBP-luciferase constructs were performed
by electroporation.
Oncogene transformation resulted in the establishment of a permanent cell line that could be readily
propagated. Immunocytochemical and immunoblotting
RESULTS.
From the Department of Ophthalmology, Northwestern University Medical School, Chicago, Illinois; the department of Pathology,
and Cell Biology and Anatomy, Morehouse Medical School, Atlanta,
Georgia; and the 3W. Alton Jones Cell Center, Lake Placid, New York.
Supported by National Eye Institute grants EY-03523 and EY06603 and by an unrestricted award from Research to Prevent Blindness Inc.
Submitted for publication January 14, 1997; revised June 10, 1997;
accepted September 19, 1997.
Proprietary interest category: N.
Reprint requests: Vijay Sarthy, Department of Ophthalmology,
Tarry 5-715, Northwestern University Medical School W113, 300 E.
Superior St., Chicago, IL 60611.
IOVS, January 1998, Vol 39, No. 1
5. Clark RA, Miller JM, Rosenbaum AL, Demer JL. Heterotopic muscie
pulleys or oblique muscle dysfunction? J Am Assoc Pediatr Ophthalmol. Stabismus. 1998. In press.
6. Guyton DL, Weingarten PE. Sensory torsion as the cause of
primary oblique muscle overaction/underaction and A- and Vpattern strabismus. Binoc Vis Eye Muscle Surg Q. 1992;9:209236.
7. Demer JL, Miller JM. Magnetic resonance imaging of the functional
anatomy of the superior oblique. Invest Ophthalmol Vis Sci. 1995;
36:906-913.
8. Miller JM, Robinson DA. A model of the mechanics of binocular
alignment. Comput Biomed Res. 1984; 17:436-470.
9. Cheng H, Burdon MA, Shun-Shin GA, Czypionka S. Dissociated eye
movements in craniosynostosis: a hypothesis revived. Br J Ophthalmol. 1993;77:563-568.
10. Krzizok T, Wagner D, Kaufmann H. Elucidation of restrictive motility in high myopia by magnetic resonance imaging. Arch Ophthalmol. 1996;115:1019-1027.
studies demonstrated that the Muller cell line, rMC-1,
expressed both GFAP, a marker for reactive gliosis in
Muller cells, and CRALBP, a marker for Muller cells in the
adult retina. Transient transfection assays showed that
promoter-proximal sequences of the CRALBP gene were
able to stimulate reporter gene expression in rMC-1.
CONCLUSIONS. Viral oncogene
transformation has been successfully used to isolate a permanent cell line that expresses Muller cell phenotype. The rMC-1 cells continue
to express both induced and basal markers found in primary Muller cell cultures as well as in the retina. The
availability of rMC-1 should facilitate gene expression studies in Muller cells and improve our understanding of Miiller cell-neuron interactions. (Invest Ophthalmol Vis Sci.
1998;39:212-2l6)
M
uller cells are the most abundant non-neuronal cells in
the vertebrate retina, and they perform diverse functions that support the activity of retinal neurons, hi
recent years, the availability of dissociated cell preparations and
primary Miiller cell cultures has greatly facilitated cell biologic,
biochemical, developmental, and electrophysiological studies of
Muller cells. Miiller cell cultures have been obtained from neonatal and adult retinas, and from retinas with inherited dystrophy or
constant light damage.1"8 However, primary Muller cell cultures
have certain problems that limit their utility: the cells have a
limited life span and undergo senescence with passage; the cultures are usually contaminated with astrocytes and microglia3'9;
unless a large number of eyes is used, only a small number of
cultures can be obtained3; and the small culture size restricts their
use to morphologic, immunocytochemical, and electrophysiological studies.
Some problems associated with primary cultures can be
overcome by establishing permanent cell lines through immortalization of primary cells with viral oncogenes. During gene
regulation studies using transfection assays, we found that the
small number of cells and the low transfection efficiency in
primary Muller cell cultures were serious drawbacks.10 This
motivated us to establish a Muller cell line. This report describes the isolation and immunochemical identification of a
Muller cell line (rMC-1) from adult rat retina.
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Reports
IOVS, January 1998, Vol 39, No. 1
METHODS
Primary Miiller cell cultures used for immortalization were
obtained from rats exposed to constant light to induce photoreceptor loss.3 In previous work, it has been reported that cell
cultures prepared from light-damaged retinas consist predominantly of Miiller cells.3 Briefly, Sprague-Dawley rats were
exposed to constant light for 2 weeks. The retinas were isolated, treated with trypsin, dissociated, and plated into 75-mm2
flasks containing Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum and glutamine. After a
single passage, primary Miiller cell cultures were transfected by
a modified calcium phosphate method."" 1 3 Ten micrograms
of simian virus 40 (SV40) DNA was mixed with 0.5 ml of 2 X
Hepes buffer, pH 7.05, and an equal volume of 0.4 M CaCl2 in
10 mM Tris, pH 7.6, and 1 mM ethylenediaminetetraacetic acid.
The mixture was applied to Miiller cells in 75-mm2 flasks
(approximately 50% confluency). After 12 hours, cells were
treated with 15% glycerol for 90 seconds at room temperature
and maintained in regular growth medium.
Clones began to appear by 2 to 3 weeks and were expanded
by repeatedly passaging cells using cloning wells as described
earlier.13 This protocol has been successfully used to obtain immortalized pigment epidielium and retinal cell lines.13 Because
our primary interest was to obtain a cell line suitable for glial
fibrillary acidic protein (GFAP) gene expression studies, seven
clones were immunostained with a GFAP antibody, and one
positive clone, rMC-1, was chosen for additional characterization.
Immunocytochemical and immunoblotting studies were carried
out as described previously.314 Animals were used according to
the tenets of the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research.
RESULTS
Figure la shows die morphology of cells from a primary culture of
Miiller cells. The majority of cells in these cultures are the large, flat
cells, which have been previously shown to be Miiller cells.3 A small
number of astrocytes, endodielial cells, or microglia are also usually
present.3'9 The Miiller cell line, rMC-1, was derived by transforming
primary cultures with SV40 DNA. Figure lb presents die morphologic features of rMC-1. Unlike the primary culaires, all cells in the
rMC-1 cultures have die same morphology—the cells are more com-
TABLE 1. Luciferase Levels in rMC-1 and 3T3 Cells
Transfected with a pCRALBP-/wc Construct
pCRALBP-/«c
pGL2B-/wc
rMC-1
3T3
180 ± 48
13.2 ± 4.1
9.8 ± 0.27
8.3 ± 0.6
Miiller cells (rMC-1) and fibroblasts (3T3) were grown to 50% to
70% confluency and cotransfected with 20 ixg of DNA per 100-mm2
culture. The construct pCRALBP-/«c contains a 0.3-kb, 5' fragment
of human CRALBP gene cloned into the promoterless vector,
pGL2B-/wc. Cotransfection with pSVlac vector was used to normalize transfection assays. Forty-eight hours following electroporation,
cells were harvested and assayed for luciferase and j3-galactosidase
using a commercially available kit. Luciferase activities were expressed as photon counts per second X 103 and were normalized to
/3-gal activities in each case to control for variations in transfection
efficiency. All values are expressed as mean ± SEM for three separate determinations.
213
pact and elongated widi a less prominent nucleus. The cells grow
rapidly with a doubling time of approximately 48 to 52 hours.
To demonstrate that rMC-1 is derived from Miiller cells, we
carried out immunocytochemical studies using antibodies to
GFAP and cellular retinaldehyde-binding protein (CRALBP). In
primary cultures, GFAP is a useful marker for reactive Miiller cells
(Fig. lc), and astrocytes, whereas endothelial cells and microglia
do not express this marker.15 CRALBP is known to be expressed
by Miiller cells but not by astrocytes in die adult mammalian
retina.16 As shown in Figures Id, le, If, rMC-1 cells were imniunoreactive for both GFAP (Fig. Id) and CRALBP (Figs, le, If).
Moreover, immunostaining was seen widi both N-temiinal (Fig.
le) and C-terminal (Fig. If) CRALBP antibodies. These results
demonstrate that rMC-1 expresses antigens specifically associated
with Miiller cells in the adult retina. An intriguing feature was that
immunostaining was often perinuclear. We do not know the
reason for this pattern of immunostaining. Although rMC-1 expressed GFAP, the intensity of immunostaining was considerably
weaker than that observed with primary cultures (compare Figs,
lc, Id). Finally, when rMC-1 cells were stained widi a monoclonal
antibody to SV40-T antigen, all nuclei were strongly reactive,
showing that rMC-1 cells contained SV40 (Fig. Ig). Omission of
the primary antibody resulted in no immunostaining (Fig. In).
In addition to immunocytochemistry, we performed immunoblotting to further characterize the antigen recognized by
GFAP and CRALBP antibodies. Cell extracts were separated by
polyacrylamide gel electrophoresis and blotted onto nitrocellulose. The blots were stained with a monospecific, polyclonal
GFAP antibody or with antipeptide, CRALBP antibodies specific to N-terminal and C-terminal regions. Extracts from primary Miiller cells or bovine retina were used as controls. As
shown in Figure 2, a single band of ~ 5 0 kDa was GFAP
immunoreactive in rMC-1 and primary Muller cell extracts,
demonstrating that rMC-1 expressed GFAP.14 In extracts from
primary cultures, there were several lower size bands that
were likely to be GFAP degradation products. Immunoblots
stained for three different CRALBP antibodies showed that, in
rat retina and rMC-1 extracts, the antibodies recognized a
single protein of about the same size as CRALBP (36 kDa). l7 An
additional band, slightly smaller in size, was noted in the cell
extracts (Fig. 2). We do not know the origin of the smaller
band; it might be a degradation product of CRALBP.
Finally, we have used rMC-1 cells to test their utility for
transfection assays. For this purpose, a construct containing
the proximal (0.3 kb), 5' sequences of the human CRALBP
gene was transfected into rMC-1 and NIH 3T3 cells, a fibroblast
cell line, which served as a control. In addition, transfections
were also carried out with a vector, pGL2B, that lacked the
CRALBP promoter. As shown in Table 1, in rMC-1, pGL2 with
a 0.3-kb CRALBP promoter stimulated luciferase expression
14-fold compared with a promoterless vector (Table 1). Moreover, the stimulation in reporter expression was cell typespecific, because luciferase activity was relatively lower in 3T3
cells. These data show that the immediate, 5'-flanking sequences of human CRALBP gene contain DNA sequences that
stimulate CRALBP gene expression in Miiller cells. Therefore,
the rMC-1 cell line allowed us to extend gene regulation studies
of CRALBP to Miiller cells, in addition to RPE cells. 1 8 1 9
DISCUSSION
The present report describes the isolation and characterization of an oncogene-transformed retinal cell line that exhib-
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214
Report
IOVS. Tanuarv 1998. Vol 39, No. 1
1. Morphology and immunocytochemical characterization of Miiller cells, (a) Primary
culture of Miiller cells, (b) Miiller cell line, rMC-1. (c) Primary Miiller cell culture stained with
glial fibrillary acidic protein (GFAP) antibody, (d) rMC-1 reacted with GFAP antibody. (e; f)
rMC-1 stained with N-terminal and C-terminal cellular retinaldehyde-binding protein antibodies, respectively, (g) rMC-1 reacted with a monoclonal antibody to SV4O T-antigen. (h) rMC-1,
immunocytochemistry with no primary antibody added. Magnifications: 160X (a, b); 960X (c,
d); 56OX (e); and 48OX (f-h).
FIGURE
its phenotypic characteristics of retinal Miiller cells. This
study shows that viral oncogene transformation can be successfully used to isolate permanent cell lines from primary
Miiller cell cultures. Our findings are consistent with a
recent report in which a disabled viral construct carrying
human papillomavirus-l6 E6/E7 genes was used to transform
Miiller cells.20 The availability of a permanently established
MiiUer cell line should facilitate a variety of studies such as
identification of Miiller cell-specific antigens, Miiller cellneuron and Muller cell-endothelial cell interactions, char-
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Reports 215
IOVS, January 1998, Vol 39, No. 1
*• f
GFAP
S17L
K25Q
*
$
Coomassie
Blue
Is
S31F
FIGURE 2. Western blot analysis of glial fibrillary acidic protein (GFAP) and cellular retinaldehyde-binding protein (CRALBP)
expression in rMC-1 cells. Extracts of rMC-1, primary Miiller cell cultures (pMC; —100 jutg), or rat retina (—100 /xg) were separated
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose, and the blots were reacted with
a polyclonal, bovine GFAP antibody (Dako, Carpinteria, CA) orpolyclonal antipeptide antibodies to bovine CRALBP. The antibodies
used for blotting are indicated underneath the blots. Closed arrow, GFAP band; open arrow, CRALBP band in the retina. The
peptide antibodies, purified immunoglobulin Gs were used at a concentration of 1 jag/ml, and recognize N-terminal CRALBP
residues 1-17 (S17L), residues 46-70 (K25Q), and Gterminal residues 286-316 (S31F). Detections were carried out with an
Enhanced Chemi-Luminescence Kit (Amersham Life Science, Arlington Heights, IL). The Coomassie Blue-stained gel following the
transfer to nitrocellulose is also shown.
acterization of growth factors and cytokines secreted by
Miiller cells, and regulation of gene expression in Miiller
cells. Moreover, these studies should lead to a better understanding of Miiller cell functions in the mammalian retina.
A cknowledgment
The authors thank Kathleen Rundell, Northwestern University, for the
monoclonal antibody to SV40-T antigen.
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